Project Summary/Abstract Recent advances in cancer immunotherapies have offered unprecedented opportunity to harness the power of the immune system to fight against cancer. Some immunotherapies, including adoptive T-cell therapy (ACT), are often preceded by chemotherapies which serve as the frontline treatment and/or host pre-conditioning regimen. The impact of chemotherapy on the efficacy of ACT is not fully understood. We previously reported that some anticancer drugs, exemplified by cyclophosphamide (CTX), can drive the expansion of myeloid cells resembling the well-studied myeloid-derived suppressor cells (MDSCs). These chemotherapy-induced MDSCs, referred to as T-iMDSCs, are able to suppress T-cell responses. We have now obtained evidence that T-iMDSCs can also induce stem-like cancer cells and enhance tumor invasion. In addition, we found that CTX treatment induces DNA methyltransferase 1 (DNMT1), and pharmacologically inactivating DNMT1 abrogates myeloid cell expansion after chemotherapy. Based on these data, we propose that T-iMDSCs emerging after chemotherapy promote the establishment of a tolerogenic tumor microenvironment (TME), which poses a significant obstacle to the efficacy of adoptive T-cell therapy. Mechanistically, we hypothesize that DNMT1 critically regulates post-chemotherapy hematopoietic recovery, giving rise to T- iMDSCs with multifaceted tumor-promoting properties. The implication of this study is that targeting T-iMDSCs relieves immunosuppression and sensitizes tumors to T cell therapy. The significance of this proposal is that it reveals a previously overlooked tumor-promoting mechanism inherent to chemotherapy. Our proposed studies will delineate the cellular and molecular mechanisms governing the generation (Aim 1), define the tumor-promoting functions of T-iMDSCs (Aim 2), establish effective strategies to target T-iMDSCs and potentiate ACT (Aim 3), and validate the presence of T-iMDSCs in patient samples. If successful, findings from this project may guide the rational design of more effective chemo-immunotherapy regimens capable of delivering durable and curative outcomes.